The following description provides a summary of information relevant to the present invention and is not an indication that any of the information provided or publications referenced herein is prior art to the presently claimed invention.
The hybridization of oligonucleotides is useful for a wide variety of analytical and diagnostic assays, including the sorting, detection, and identification of analytes. It is important that the non-complementary oligonucleotides do not cross-hybridize with one another in such assays. Cross-hybridization of oligonucleotides is in part due to the variability in base-stacking energies among the different nucleotides and nucleotide sequence variations within oligonucleotides. Another set of parameters effecting oligonucleotide cross-hybridization is the hybridization reaction conditions. Unfortunately, such non-specific cross-hybridization is a common problem and is the primary cause of false positive or false negative signals in oligonucleotide-based assays, in particular where multiple oligonucleotide pairs are employed.
Other factors that pose problems to the design and implementation of an assay system utilizing multiple oligonucleotides are the desirability that substantially all of the paired oligonucleotides have substantially the same Tm, that all paired oligonucleotides hybridize at substantially the same rate and within the same time frame, and the desirability that the oligonucleotides hybridize together relatively rapidly at ambient temperatures without significant cross-hybridization.
What is needed is an oligonucleotide-based assay system useful for the detection of multiple analytes that utilizes a set of oligonucleotide pairs having no significant cross-hybridization between different pairs, and no consequent false positive or negative signals. A need also exists for an oligonucleotide-based assay system where all paired oligonucleotides hybridize at substantially the same rate, have substantially the same Tm, and where complementary oligonucleotide pairs are able to hybridize together relatively rapidly at ambient temperatures with no significant cross-hybridization. Unfortunately, to date such an assay system has been unavailable.